Anti ldlr

Human monoclonal antibodies AR3A, AR4A, and AR5A against HCV structural proteins were produced as described previously [27 (link);33 (link)]. Antibodies against HCV receptors were anti-CD81 (BD Pharmingen Cat. JS81), anti-SR-BI C16-17 [67 (link);77 (link)] and polyclonal anti-LDLr (R&D Systems Cat. AF2148). Control antibodies for receptor blocking assays were the isotype antibody 553447 for CD81, the antibody D for SR-BI [77 (link)], and a goat IgG (R&D Systems Cat. AB-108C) for LDLr. Soluble CD81 protein was a kind gift from Steven Foung [73 (link)]. Adapted recombinants with the core-NS2 sequence from genotypes 1a (H77), 2a (J6), 3a (S52), 4a (ED43), 5a (SA13) and 6a (HK6a) and UTR´s as well NS3-NS5B region from genotype 2a (JFH1) with or without HVR1 were described previously [44 (link);54 (link);58 (link)–61 (link)]. H77 E1E2 expression plasmids, previously validated for functionality in HCVpp assays [67 (link)], were used for E1/E2 interaction studies. Plasmids with point mutations were generated by conventional cloning techniques (Fusion PCR and Quikchange). The HCV sequence of the final plasmid DNA preparations were confirmed by direct sequencing (Macrogen). Antibody against NS5A, 9E10 [61 (link)], was provided by Charles Rice.

All reagents were obtained from Sigma unless stated otherwise. Human plasma low density lipoprotein (LDL) was obtained from Calbiochem (Gibbstown, NJ, USA). The antibodies used were the following: anti-LDLR from R&D systems, anti-VE-Cadherin from Santa Cruz, anti-ZO-1 from Invitrogen, anti-Caveolin-1 from Santa Cruz and anti-Clathrin Heavy Chain from BD Biosciences. Alexa Fluor 488 secondary antibody was obtained from Invitrogen and VECTASHIELD mounting media from Vector Laboratories. HRP-conjugated secondary antibodies were purchased from Vector Laboratories and on-target plus siRNAs from Dharmacon.